1. Field of the Invention
The present invention generally relates to microfluidic and nanofluidic analysis of analytes. In particular, the present invention relates assaying analytes based on their mobilization characteristics in applied mobilization fields.
2. Description of the Related Art
Separation by size or mass is a fundamental analytical and preparative technique in biology, medicine, chemistry, and industry. Conventional methods include gel electrophoresis, capillary electroosmotic chromatography (CEC), field-flow fractionation, sedimentation and size exclusion chromatography (SEC).
A few microfluidic and nanofluidic devices for separation and analysis of biomolecules and compounds have been developed. U.S. Pat. No. 5,427,663 discloses separating nucleic acid molecules using electric fields through an array of posts as sieving matrices. Chou et al. (Proceedings of the National Academy of Science, USA, 1999, v. 96:13,762) disclose sorting nucleic acid molecules according to their diffusion coefficients using an electric field which propels the molecules through gaps formed by an asymmetric array of objects. Han and Craighead (Science, 2000, v. 288:1026-1029) disclose separations using entropic traps consisting of a series of many narrow constrictions <<100 nm) separated by wider and deeper regions (a few microns). Huang et al. (Nature Biotechnology, 2002, v. 20:1048) disclose a hexagonal array of posts which act as a sieving matrix in pulsed-field electrophoresis. Published U.S. Patent Application 20040144651 discloses an array of obstacles wherein molecules are separated according to size. Unfortunately, the arrays required for providing patterned field non-uniformities suitable for assaying analytes is costly and impracticable for processing real world samples.
Similarly, various attempts to assay intact bacteria viruses in real world samples using capillary electrophoresis (alone) had limited success due to the relative low concentration of cells in the samples. See Armstrong et al. Analytical Chemistry, 1999, v. 71:5465, and Armstrong et al. Analytical Chemistry, 2001, v. 73:4551. Methods to select and/or concentrate cells are needed to improve detection sensitivity in real world samples.
Dielectrophoresis, on the other hand, enables trapping and concentration of particulates in non-uniform electric fields. For example, the Washizu group in Japan used arrays of embedded electrodes to separate latex particles (100 nm and 1 μm in diameter) and DNA particulates purely based on dielectrophoretic effects. See Sano et al. Institute of Electrical and Electronics Engineers, Transactions of Industry Applications, 2002, v. 11. The Washizu group calls this technique dielectrophoretic “chromatography”; however, the separation is solely due to dielectric effects and no chromatography, i.e. adsorption, desorption mechanism, is involved.
Lin et al. (Journal of Electrostatics, 1982, v. 13:257) use dielectric filtration and high-gradient electric separation induced by a dielectric body (e.g. ceramic beads) in a uniform electric field to remove particulates in large-scale industrial applications suitable for liquids with low conductivity. Suchiro et al. (Institute of Electrical and Electronics Engineers, Transactions of Industry Applications, 2003, v. 39:1514) use two parallel plate electrodes to create non-uniform electric field gradients. Se also Zhou et al., Institute of Electrical and Electronics Engineers, Transactions of Industry Applications, 2002, v. 2:1404.
Unfortunately, devices with embedded electrodes or devices with dielectric particulates are difficult, expensive and hard to manufacture reproducibly especially for microliter volume applications. In addition, integration in a complex multifunctional system is challenging due to inherent band broadening effects that occur during transition from chip to other processing and/or detection devices. Due to the small size of microfluidic devices, localized surface modification with application specific chemical groups is extremely difficult, as is the control of surface porosity. Such limitations on surface characteristics render embedded electrode-based devices unsuitable for chromatographic applications and nanodielectrophoretic experiments.
Kreibik et al. (Journal of Planar Chromatography, 2001, v. 14:246) introduced a technique, “planar dielectrochromatography”, which uses non-linear effects generated by alternating currents to pump liquids through a planar thin layer of chromatographic plate to developed distinctive spots of dyes; the plate system has very low sensitivity and has limited applicability as it is not suitable for high-throughput high-sensitivity methods required in bioanalysis.
There is therefore a need for economical microfluidic devices and methods which are easy to fabricate and use for rapidly processing and assaying particulates in fluid samples.